INTRODUCTION:

Curettage, a periodontal surgical treatment, is employed when initial therapies like scaling and root planing (SRP) prove less effective or have limited access to deep subgingival areas. Unfortunately, this limitation allows certain pathogens such as Actinobacillus actinomycetemcomitans, Porphyromonasgingivalis, Prevotella intermedia, Bacteroides forsythus, and Peptostreptococci micros—to persist¹^,²,³^,⁴.

In curettage, the gingival wall within a periodontal pocket is scraped to remove chronically inflamed tissue². Additionally, curettage aims to promote root attachments and form periodontal pocket walls that procedure involve local anesthesia, scraping, and irrigation⁵.

During curettage healing, the scrapping of periodontal pocket walls aims to eliminate pockets, repair attachments, or create new attachments. However, this action will cause injury because the tissue substance is damaged or missing. The wound healing process involves several distinct phases: the inflammatory phase, proliferation phase, and maturation phase. During the proliferation phase of the wound healing process, fibroblast cells play a crucial role during the proliferation phase and contribute to tissue repair⁶,⁷.

When a wound occurs, the main treatment that must be carried out is debridement, irrigation, and the administration of antibiotics or antiseptics⁶. Irrigation in curettage treatment aims to eliminate bacteria and reduce the development of gingivitis. Based on this, materials that are needed for irrigation in curettage should be able to inhibit the growth of periopathogenic bacteria, accelerate fibroblast proliferation, and not be cytotoxic to fibroblasts³^,⁸^,⁹^,¹⁰.

Aloe vera has demonstrated effective antibacterial activity in inhibiting the growth of periopathogenic bacteria. Active components of aloe vera, which play a role in antibacterial activity, come from the content of flavonoids, saponins, tannins, and alkaloids¹¹,¹²^,¹³. Flavonoids can cause damage to bacterial cell membranes by inhibiting cell membrane function and bacterial metabolism. Saponins have antibacterial capabilities because they can destroy bacterial cells by disrupting the permeability of bacterial cell membranes and releasing several vital components from bacteria. The mechanism of tannin as an antibacterial is to inactivate bacterial adhesins, inhibit enzyme action, and inhibit protein transport in cell membranes¹⁴.

Aloe vera solution has also been proven to speed up wound healing after scaling and is effective in healing wounds by increasing the number of fibroblast cells¹⁵^,¹⁶. Antibacterial activity and its role in wound healing make aloe vera extract an alternative sustainable curettage irrigation material. Before being used as an alternative dental material, the toxicity of aloe vera extract must be tested. This is done because the materials used in dentistry must have good biocompatibility. The initial stage in testing the biocompatibility of a material is an in vitro cytotoxicity test in which the material to be tested is contacted outside the body of the microorganism, such as cell culture¹⁷.

This cytotoxicity test was carried out using the Microculture Tetrazolium Technique assay (MTT assay) method with gingival fibroblast cell culture as a test medium. The MTT Test Assay is a colorimetric test to measure cell growth that can be used to assess the toxicity of toxic substances. MTT assay based on measurements of the mitochondrial activity of living cells. MTT assay uses a water-soluble yellow dye, namely tetrazolium salt, which is easily absorbed by living cells. Within living cells, tetrazolium salt undergoes reduction to form purple formazan. This reduction process occurs due to the action of the enzyme succinate dehydrogenase in the mitochondria¹⁸. This causes cells that are still alive and whose metabolism is active to change the tetrazolium salt, which was originally yellow, into a purple formazan product. This purple formazan crystal product is quantified using a spectrophotometer. The amount of formazan produced directly correlates with the number of viable cells in the sample¹⁸,¹⁹^,²⁰.

The MTT assay was selected because it can be used to measure large quantities of samples relatively quickly, sensitively, and accurately²¹. By assessing the linear relationship between metabolically active cells and the resulting color, the MTT assay allows precise quantification of changes in cell death or proliferation rates¹⁹. The (MTT assay) has long been considered the gold standard²².

The cells used as test media in this study were primary cell cultures derived from human gingival fibroblast cells. Gingival fibroblast cells are the most abundant cell type that can be found in periodontal and gingival connective tissue². Based on their regeneration ability, fibroblast cells are one of the stable cells. Stable cells have minimal proliferation activity under normal conditions. However, these cells can divide in response to injury^21,22. Fibroblast cells are responsible for preparing and producing structural protein products that are used during the process of tissue repair or reconstruction⁶. Human gingival fibroblast cells were chosen for the cytotoxicity test because they have a higher level of sensitivity when compared with other fibroblast cells²³.There is no research regarding the cytotoxicity of aloe vera, which made the author interested in conducting a cytotoxicity test of aloe vera extract with concentrations of 10.5%, 11.5%, 12.5%, 13.5%, and 14.5%.

MATERIALS AND METHODS:

Materials:

The study utilized healthy human gingival fibroblast cells obtained after odontectomy in January 2024. These cell preparations belong to the Research Center of Dental Medicine Laboratory, Faculty of Dentistry, Airlangga University, Surabaya. The investigation involved three distinct groups: namely the positive control group: comprising gingival fibroblast cells, the negative control group consisting of cell culture media Dulbecco’s modified eagle medium (DMEM), and the treatment group consisting of gingival fibroblast cells treated with aloe vera extract with concentrations of 10.5%, 11.5%, 12.5%, 13.5%, and 14.5%.

Methods:

Aloe vera extract is a preparation of aloe vera in the form of a 100% thick extract obtained from a maceration process using 70% ethanol solvent. The aloe vera extract was then made into several different concentrations, namely 10.5%, 11.5%, 12.5%, 13.5%, and 14.5%, using DMEM cell media solvent. (Dulbecco’s modified eagle medium).

Cell culture is divided into 96 microplate wells of 100 μl with a density of 3x10³. Fibroblast cell culture was divided into 7 groups (wells 1–7) with each group having 8 wells, then incubated at a temperature of 37 °C for 24 hours. Before treatment, the fibroblast cells in the microplate that had been incubated were observed under a light microscope to ensure that there were enough fibroblast cells planted in each well for research. Wells in columns 1–5 were treated with DMEM culture media and exposed to aloe vera extract according to the concentration of 50μl and repeated 8 times for each well. Wells 6 and 7 were treated according to the control group.

To perform the tetrazolium salt assay, dissolve the tetrazolium salt in phosphate-buffered saline (PBS) at a concentration of 5mg/mL. Add 25μL of this solution to each well of the microplate. Incubate the plate for 4 hours at 37°C. Afterward, discard all the DMEM culture media in the microplate and add 100μL of dimethyl sulfoxide (DMSO) to each well.

Then observe the condition of the gingival fibroblast cells under a microscope, observe the color changes that occur on the microplate after treatment, and determine the value of the optical density obtained from reading the absorbance of formazan crystals (OD) in human gingival fibroblast cells spectrophotometrically using an ELISA reader with a wavelength of 540nm. The OD value obtained is entered into the cell viability formula (% live cells) to determine whether or not aloe vera is toxic to gingival fibroblast cells.

The cell viability formula (% live cells):

OD treatment – OD media

Cell Viabillity = ---------------------------------- X 100%

(% live cells) OD cell – OD media

Information:

· Cell Viability (% Live Cells): Percentage of the number of cell life after the test

· OD Treatment: Formazan Optical Density (OD) value of each sample after the test

· OD Cell: Formazan OD value on the mean of control cells

· OD Media: Formazan OD value on the average of each control media

The data obtained was analyzed statistically through software. The statistical test used is the normality test (Kolmogorov-Smirnov). We assessed data homogeneity using the Levene test. If the obtained data is both normally distributed and homogeneous (p>0.05), we proceed with the parametric one-way ANOVA to examine the effect of human gingival fibroblast cell viability among treatment groups. Conversely, if the data is not normally distributed and lacks homogeneity (p<0.05), we employ the non-parametric Kruskal-Wallis test to identify differences in the percentage of living cells across research groups. Subsequently, we conduct the Tukey HSD test. By using this method, the researcher can find the differences that occur between each treatment group.

RESULT:

The results obtained from this research include observations of the condition of gingival fibroblast cells under a microscope before and after treatment, color changes in the microplate following treatment, and optical density (OD) readings using an ELISA reader.

Figure 1. Microscopic observations of gingival fibroblast cells as a Cell Control (A) Gingival fibroblast cells before treated with tetrazolium salt (B) Gingival fibroblast cells after treated with tetrazolium salt

Figure 2: Microscopic observations of media Control DMEM (A) Gingival fibroblast cells before treated with tetrazolium salt (B) Gingival fibroblast cells after treated with tetrazolium salt

Figure 3. Microscopic observations of gingival fibroblast cells treated with aloe vera extract concentration 10.5% (A) Gingival fibroblast cells before treated with tetrazolium salt (B) Gingival fibroblast cells after treated with tetrazolium salt

Figure 4. Microscopic observations of gingival fibroblast cells treated with aloe vera extract concentration 11.5% (A) Gingival fibroblast cells before treated with tetrazolium salt (B) Gingival fibroblast cells after treated with tetrazolium salt

Figure 5. Microscopic observations of gingival fibroblast cells treated with aloe vera extract concentration 12.5% (A) Gingival fibroblast cells before treated with tetrazolium salt (B) Gingival fibroblast cells after treated with tetrazolium salt

Figure 1. Microscopic observations of gingival fibroblast cells treated with aloe vera extract concentration 13.5% (A) Gingival fibroblast cells before treated with tetrazolium salt (B) Gingival fibroblast cells after treated with tetrazolium salt

Figure 7. Microscopic observations of gingival fibroblast cells treated with aloe vera extract concentration 14.5% (A) Gingival fibroblast cells before treated with tetrazolium salt (B) Gingival fibroblast cells after treated with tetrazolium salt

Microscopic observations of gingival fibroblast cells treated with tetrazolium salt in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, and Figure 7 reveal the formation of formazan crystals in the form of black dots. These crystals indicate that the gingival fibroblast cells can still survive after treatment. Conversely, in dead gingival fibroblast cells, no black dots are present, signifying the absence of formazan crystal formation. The media control group (negative control) did not exhibit any black dots after treatment because no fibroblast cells were present in this group. Notably, the greater the number of black dots observed, the more viable cells are present. Additionally, the images demonstrate that higher concentrations result in fewer black dots. Figure 8 shows the color changes that occur in the microplate after treatment; it becomes purplish-blue. The color change to purplish-blue indicates that the material tested is not toxic to gingival fibroblast cells. The color density after administration of tetrazolium salt indicates the percentage of living cells. The intensity or density of the formazan color is directly proportional to the number of living cells. The darker the purplish-blue color that appears after the administration of tetrazolium salt, the higher the percentage of living cells. The control group of cells had a higher concentration of purplish blue than the other groups because the control group only contained cells, where it was assumed that the percentage of living cells (cell viability) was 100%.

Figure 8. Formazan color intensity after treated with tetrazolium salt in Microplate 96 Well

Figure 8 shows that the formazan color intensity of the extract treatment group with concentrations of 10.5%, 11.5%, 12.5%, 13.5%, and up to 14.5% decreased. The lower the concentration of the extract, the darker the color that appears, but the intensity or density of the color that appears in the treatment group is very low compared to the control cell group. The media control group did not show any color changes because there were no cells in that group.

After the two observations above have been carried out, continue reading the OD value after treatment using the ELISA reader. The OD value obtained is then entered into the formula for the percentage of living cells (cell viability) to determine whether the test material is toxic to gingival fibroblast cells. The results of OD and cell viability can be seen in the following table.

Results from Table 1, show that there has been a change in the average OD value and the standard deviation for each group. The data in the table shows that the higher the extract concentration, the lower the average OD value and cell viability. Low average OD and cell viability values indicate a low level of live cells (high dead cells).

The highest cell viability value in the treatment group, namely 84.5753%, was found in aloe vera extract with a concentration of 10.5%. Meanwhile, the lowest cell viability in the treatment group, namely 65.8387%, was found in aloe vera extract with a concentration of 14.5%. The media control group is assumed to have a viability percentage value of 0%, while the cell control group The toxicity level of a material, based on the percentage of living cells, can be classified according to Heravi as follows: can be classified into non-toxic (> 90% living cells), slightly toxic (60–90% living cells), toxic (30–59% living cells), and very toxic (viable cells < 30%).In this study, the percentage of viable cells fell within the 60–90% range (slightly toxic) at concentrations of 10.5%, 11.5%, 12.5%, 13.5%, and 14.5%. This indicates a slightly toxic effect on human gingival fibroblast cells.

The data were analyzed using analytical statistical tests in the SPSS 27.0 program, a Kolmogorov-Smirnov test was conducted to assess data normality. Based on a sample size of 56, the data were found to be normally distributed (p > 0.05). Continuing with the analysis, we assessed the homogeneity of the data using Levene’s test (p > 0.05). However, the result indicated non-homogeneous data (p = 0.001, p < 0.05). Despite this, we concluded that the existing data remained normally distributed.Next, we conducted significance tests using non-parametric statistical methods, specifically the Kruskal-Wallis test, to identify significant differences among the treatment groups. The result demonstrated a significant difference in the treatment group (p = 0.001, p < 0.05).

To further explore differences between each treatment group, we performed a Tukey HSD test. Significant differences between groups are evident when the significance results fall below 0.05 (p < 0.05). Conversely, if the significance results exceed 0.05 (p > 0.05), we conclude that no significant differences exist between treatment groups.

DISCUSSION:

Materials used in dentistry must meet biocompatibility requirements, that is, perform their function without causing undesirable effects. The initial stage in testing the biocompatibility of a material is a cytotoxicity test in vitro¹⁷. In this study, a cytotoxicity test was carried out in vitro with aloe vera extract at concentrations of 10.5%, 11.5%, 12.5%, 13.5%, and 14.5% against gingival fibroblast cells using the Microculture Tetrazolium Technique assay (MTT assay).

The MTT test assay is a colorimetric test used to measure cell growth and assess the toxicity of various substances. This method relies on measuring the mitochondrial activity of living cells. In the MTT assay, a water-soluble yellow dye—specifically, tetrazolium salt—is introduced, which living cells readily absorb. Cells that remain alive and metabolically active can convert the initially yellow tetrazolium salts into purple formazan products¹⁸^,¹⁹^,24.

The reduction of tetrazolium salt to purple formazan occurs through the action of the enzyme succinate dehydrogenase in the mitochondria of living cells¹⁸^,25. When a material is non-toxic, the dehydrogenase remains active, resulting in the production of formazan crystals. These crystals cause fibroblast cells to stain purplish-blue. In contrast, cells with membrane damage do not exhibit staining. Notably, the intensity of the purplish-blue color following tetrazolium salt administration correlates with the percentage of living cells¹⁹.

The percentage of human gingival fibroblast cells that are still alive after treatment can indicate the level of cytotoxicity of each sample group²⁴. The level of toxicity of a material based on the percentage of living cells, according to Heravi, can be classified into non-toxic (> 90% living cells), slightly toxic (60–90% living cells), toxic (30–59% living cells), and very toxic (viable cells < 30%)²⁶. The results of the study showed that aloe vera extract with concentrations of 10.5%, 11.5%, 12.5%, 13.5%, and 14.5% has a slightly toxic effect on human gingival fibroblast cells because the viable cells are in the range of 60–90%.

The research results reveal that the average optical density (OD) value and cell viability decrease as the extract concentration increases. Lower average OD and cell viability values correspond to a higher proportion of dead cells. Consequently, greater influence leads to increased cell death, resulting in a higher percentage of cell mortality²⁷. Several biochemical motifs are thought to mediate cell death; this can explain the occurrence of cell death in connection with the cytotoxicity of a material²⁸.

These biochemical motifs include the depletion of ATP (Adenosine Triphosphate) levels. The dehydrogenase enzyme is one of the enzymes that plays a role in the formation of ATP, a form of energy that cells need for various functional cell activities. If the dehydrogenase enzyme is inactive due to the cytotoxic effects of a cytotoxin, then ATP is reduced and cell activity is disrupted, which can result in cell death²⁸. Apart from that, defects in the cell membrane can also cause cell death. Membrane damage or loss of selective membrane permeability is a common feature of cell injury. This defect can affect the mitochondria, which are the place for producing ATP²⁸.

The functional activity of gingival fibroblast cells, namely proliferating and producing extracellular matrix and fibronectin, in connection with the gingival tissue repair process, will be disrupted if exposure to cytotoxins causes the cells to experience irreversible injury or results in non-viable cells⁶^,²⁸. This is because all cellular mechanisms are interrelated processes; if one cell component is injured, then all cell activities will be affected, including the tissue repair process, which requires the regeneration of cells that have experienced injury. Cells remain viable if exposure is not cytotoxic, so cell functional activity will not be disrupted²⁸.

General principles regarding cell injury that may be the cause of differences in effects in the research groups include the existence of several components or intracellular systems of cells that are sensitive or easily injured, such as cell membranes (cell membrane integrity), aerobic respiration systems (mitochondria, enzymes), and genetic components. In addition, the cellular response to injury stimuli depends on the type of injury, the duration of the injury stimulus, and the severity of the injury stimulus. Types of injury are differentiated based on the cause of cell injury, such as chemicals and drugs, hypoxia, and others. The length of the stimulus injury is related to the exposure time, while the severity of the stimulus is related to the dose or concentration of exposure²⁸.

There is a correlation between the concentration of the test solution and its cytotoxicity. As the concentration increases, the number of cells that die increases. This means that the higher the concentration of the test solution, the lower the absorbance of the wells so that the percent of life becomes smaller²⁷. This increasingly small percentage of life is supported by microscopic observations where gingival fibroblast cells are seen secreting formazan crystals in appropriate amounts. This indicates that aloe vera extract with concentrations of 10.5%, 11.5%, 12.5%, 13.5%, and 14.5% is slightly toxic, but gingival fibroblast cells can still survive even in relatively small amounts.

The active components of aloe vera include flavonoids, saponins, tannins, and alkaloids (about aloe vera). Several active components that are present in aloe vera can be cytotoxic to gingival fibroblast cells, causing cell death.Active compounds can be toxic at high doses²⁹. Flavonoids are a secondary metabolite that will be toxic if given in excess. At low concentrations, flavonoids act as antioxidants by capturing free radicals, thereby preventing cell oxidation and protein denaturation²⁹. Increasing the concentration of aloe vera extract increases the antioxidant properties of flavonoids and converts them into prooxidants, which can trigger the formation of reactive oxygen species (ROS), thereby causing oxidative stress. It can also inhibit cell proliferation, inactivate DNA oxidation, and produce free radicals that cause cell lysis²⁷.

As an active secondary metabolite compound, tannin is a polyphenol derivative with a high level of polarity; therefore, when given in high concentrations, it can cause genotoxicity, namely damage to genetic information in cells. The presence of bonds between polar compounds and cell lipoproteins can result in the accumulation of compounds and the breakdown of fat, so that the permeability of periodontal ligament fibroblast cells will be disrupted, causing cell necrosis²⁷.

Saponins are a group of glycosides that are found in many plants, including aloe vera In saponin compounds. These compounds consist of amphipathic molecules with both hydrophilic and hydrophobic regions. When the hydrophobic end of a saponin molecule binds to the hydrophobic region of cell membrane proteins, it disrupts the lipid elements within the membrane. Simultaneously, the hydrophilic end of the saponin forms a detergent-protein complex, leading to cell membrane rupture and lysis, ultimately resulting in cell necrosis. Additionally, saponins can induce apoptosis in fibroblast cell cultures²⁵.

Alkaloids work specifically on the cell cycle by inhibiting the mitosis process. The presence of obstacles to cell division can cause the cell proliferation process to be hampered. Alkaloid compounds can also cause binding to tubulin by inhibiting protein polymerization into microtubules. As a result, the function of tubulin as a cytoskeleton (building up the shape and skeleton of cells) is disrupted, resulting in disruption of the transport of substances into cells²⁶.

CONCLUSION:

Aloe vera extract with a concentration of 10.5% still had a slightly toxic effect on human gingival fibroblast cells because cell viability did not reach 90%. Consequently, this concentration is not potentially suitable as an alternative irrigation material for curettage treatment. Additionally, it is essential to investigate the cytotoxic effects of aloe vera at concentrations below 10.5%.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 25.03.2024 Revised on 03.05.2024

Accepted on 10.07.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):305-311.

DOI: 10.52711/0974-360X.2025.00047

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

	Media Control	Cell Control	14,5%	13,5%	12,5%	11,5%	10,5%
Average OD	0.05438	0.462	0.32275	0.341	0.361	0.38475	0.39913
Cell Viability (%)	0	100	65.8387	70.3159	75.2223	81.0488	84.5753
Standard Deviation	0,00256	0,06852	0,05146	0,05741	0,03594	0,06099	0,05138

Groups	10,5%	11,5%	12,5%	13,5%	14,5%	Media Control	Cell Control
10,5%		0,998	0,784	0,277	0,062	0.000	0,197
11,5%	0,998		0,966	0,612	0,210	0,000	0,057
12,5%	0,784	0,966		0,986	0,746	0,000	0,004
13,5%	0,277	0,612	0,986		0,991	0,000	0,000
14,5%	0,062	0,210	0,746	0,991		0,000	0,000
Media Control	0.000	0.000	0.000	0.000	0.000		0.000
Cell Control	0,197	0.057	0.004	0.000	0.000	0.000